Neurogenesis begins with the patterned specification of neural precursor cell fates within ectodermal tissue, a process controlled by the proneural proteins, a family of basic helix-loop-helix transcriptional activators. This function of the proneural factors originated deep in metazoan evolutionary history and is highly conserved. Understanding the process of neural precursor determination will require the identification and elucidation of (1) the cis-regulatory modules through which proneural proteins direct neural precursor-specific gene expression, and (2) the mechanisms by which proneural gene and protein activities are controlled. The research program proposed here seeks to address both of these fundamental questions. Our project has three major objectives, all focusing on the transcriptional specification of neural precursor cell fates in Drosophila: Specific Aim 1. Investigate modes of negative regulation of proneural gene activity. Published studies and recent unpublished investigations in our laboratory have demonstrated that the Drosophila proneural genes achaete and scute are subject to multiple modes of negative regulation. We will first refine our definition of the relevant gene motifs and protein domains, and then investigate how each of these sequences functions. Specific Aim 2. Elucidate the roles of dual cis-regulatory modules in sensory organ development. Our recent studies have revealed that several genes expressed specifically or predominantly in sensory organ precursor cells in Drosophila rely on at least two transcriptional cis-regulatory modules of overlapping specificity to direct their activity in these cells. We will identify such dual enhancers systematically in a selected set of neural precursor genes, and investigate how each module contributes to the expression and phenotypic function of the associated gene. Specific Aim 3. Evaluate a proposed transcriptional cis-regulatory code for expression in neural precursor cells. Our laboratory has identified in a variety of Drosophila genes transcriptional cis-regulatory modules with specificity for sensory organ precursor cells. Out of these studies has come the hypothesis that a critical subset of these modules utilize a specific cis-regulatory "code" (a combination of binding sites for proneural activators and basic helix-loop-helix repressors) to confer neural precursor-specific expression. We will test this hypothesis, and investigate how such modules contribute to proper neural precursor specification. A broad spectrum of human tumors with a "neuroendocrine" phenotype are associated with strong overexpression of proneural genes, including small cell lung cancer (the most lethal form of lung cancer), neuroblastoma (an aggressive childhood cancer), medullary thyroid cancer, pheochromocytoma (a tumor of the adrenal medulla), small cell neuroendocrine carcinoma of the prostate, and Merkel cell carcinoma of the skin. By elucidating mechanisms that normally regulate proneural gene activity negatively, our work may offer important insight into the pathogenesis of these diseases. PUBLIC HEALTH RELEVANCE: The development of the human nervous system begins with the designation of certain cells as "neural precursor cells", a process that is under the control of the "proneural" genes. Over the years we have come to realize that a broad spectrum of human tumors are associated with hyperactivity of these proneural genes, including small cell lung cancer (the most lethal form of lung cancer), neuroblastoma (an aggressive childhood cancer), medullary thyroid cancer, pheochromocytoma (a tumor of the adrenal gland), small cell neuroendocrine carcinoma of the prostate, and Merkel cell carcinoma of the skin. By studying the mechanisms that normally act to hold proneural gene activity in check, our work may offer important insight into the genesis of these diseases.